Nondestructive, High-Resolution T Cell Characterization and Subtyping via Deep-UV Microscopy.

T cell characterization is critical for understanding immune function, monitoring disease progression, and optimizing cell-based therapies. Current technologies to characterize T cells, such as flow cytometry, require fluorescent labeling and typically are destructive endpoint measurements. Non-destructive, label-free imaging methods have been proposed, but face limitations with throughput, specificity, and system complexity. Here we demonstrate deep-ultraviolet (UV) microscopy as a label-free, non-destructive, fast and simple imaging approach for assessing T cell viability, activation state, and subtype with high accuracy. Using static deep-UV images, we characterize T cell viability and activation state, demonstrating excellent agreement with flow cytometry measurements. We further apply dynamic deep-UV imaging to quantify intracellular activity, enabling fast and accurate subtyping of CD4 + and CD8 + T cells. These results corroborate recent studies on metabolic activity differences between these subtypes, but now with deep-UV microscopy they are enabled by a non-destructive, fast, low-cost and simple approach. Together, our results demonstrate deep-UV microscopy as a powerful tool for high-throughput immune cell characterization, with broad applications in immunology re-search, immune monitoring, and development of emerging cell-based therapies.

Multi-omic profiling of tumor-infiltrating T cells provides new insights into the differences in the effectiveness of CDK4 inhibitor between human papillomavirus (HPV)-positive and HPV-negative head and neck squamous cell carcinoma patients.

Multi-omic profiling of tumor-infiltrating T cells provides new insights into the differences in the effectiveness of CDK4 inhibitor between human papillomavirus (HPV)-positive and HPV-negative head and neck squamous cell carcinoma patients.

Multi-omic profiling of tumor-infiltrating T cells provides new insights into the differences in the effectiveness of CDK4 inhibitor between human papillomavirus (HPV)-positive and HPV-negative head and neck squamous cell carcinoma patients.

Recent research suggests higher circulating lymphocyte counts may protect against colorectal cancer (CRC). However, the role of specific lymphocyte subtypes and activation states remain unclear. CD4+ T cells—a highly dynamic lymphocyte subtype—undergo gene expression changes upon activation that are critical to their effector function. Previous studies using bulk tissue have limited our understanding of their role in CRC risk to static associations. We applied Mendelian randomization (MR) and genetic colocalisation to evaluate causal relationships of gene expression on CRC risk across multiple CD4+ T cell subtypes and activation states. Genetic proxies were obtained from single-cell transcriptomic data, allowing us to investigate the causal effect of expression of 1,805 genes across CD4+ T cell activation states on CRC risk (78,473 cases; 107,143 controls). Analyses were stratified by CRC anatomical subsites and sex, with sensitivity analyses assessing whether the observed effect estimates were likely to be CD4+ T cell-specific. We identified 6 genes—FADS2, FHL3, HLA-DRB1, HLA-DRB5, RPL28, and TMEM258—with strong evidence for a causal role in CRC development (FDR-P < 0.05; colocalisation H4 > 0.8). Causal estimates varied by CD4+ T cell subtype, activation state, CRC subsite and sex. However, many of genetic proxies used to instrument gene expression in CD4+ T cells also act as eQTLs in other tissues, highlighting the challenges of using genetic proxies to instrument tissue-specific expression changes. We demonstrate the importance of capturing the dynamic nature of CD4+ T cells in understanding CRC risk, and prioritize genes for further investigation in cancer prevention.

Using a high-throughput mitochondrial phenotyping platform to quantify multiple mitochondrial features among molecularly defined immune cell subtypes, we quantify the natural variation in mitochondrial DNA copy number (mtDNAcn), citrate synthase, and respiratory chain enzymatic activities in human neutrophils, monocytes, B cells, and naïve and memory T lymphocyte subtypes. In mixed peripheral blood mononuclear cells (PBMCs) from the same individuals, we show to what extent mitochondrial measures are confounded by both cell type distributions and contaminating platelets. Cell subtype-specific measures among women and men spanning four decades of life indicate potential age- and sex-related differences, including an age-related elevation in mtDNAcn, which are masked or blunted in mixed PBMCs. Finally, a proof-of-concept, repeated-measures study in a single individual validates cell type differences and also reveals week-to-week changes in mitochondrial activities. Larger studies are required to validate and mechanistically extend these findings. These mitochondrial phenotyping data build upon established immunometabolic differences among leukocyte subpopulations, and provide foundational quantitative knowledge to develop interpretable blood-based assays of mitochondrial health.

Introduction: The innate immune system profoundly influences cancer development as well as its response to various therapeutic approaches. Multiple myeloid cell lineages are central to the innate immune response during cancer and have been found to regulate diverse cellular processes encompassing immune suppression, angiogenesis, invasion, metastasis, and drug resistance. NanoString has collaborated with the laboratory of Prof. Lisa M. Coussens to develop research gene panels for quantitative and qualitative analysis of various myeloid subsets by evaluating relative enrichment of genes that mark recruitment and presence of select myeloid subtypes (e.g., granulocytes, dendritic cells, monocytes, macrophages, mast cells), their cell function, activation and effector status. Methods: Gene content for human and murine versions of the myeloid panels were developed from an in-house high content proteomic screen of macrophage activation (unpublished), genomic analysis of human tumor associated macrophages (unpublished), and data mining from publically available datasets featuring myeloid transcriptomes. In total, 692 genes were included in the human panel and 675 genes in the murine panel. The panels were optimized to work across a range of sample types including fresh frozen tissues, formalin-fixed paraffin-embedded (FFPE) samples, peripheral blood mononuclear cells and cell lysates. Results: To validate the panel content, we are profiling myeloid infiltrates in syngeneic murine models of mesothelioma and mammary tumorigenesis by flow cytometric analysis and the nCounter myeloid panels. Since the quality of the myeloid tumor response can skew toward adaptive immune activating (Th1) or suppressive (Th2) phenotypes, we will profile the same tumor samples with the NanoString PanCancer Immune Profiling panel to evaluate T cell activation status by gene expression and flow cytometry as well as global immune response. Finally, myeloid complexity and T cell activation status in both primary tumor and lung metastases will be evaluated by NanoString to assess regulation of adaptive immune responses by the innate compartment in distinct tumor microenvironments. Conclusions: Upon validation with these assays, the myeloid gene panels may provide insight into how the innate immune system regulates cancer development and response to therapeutic pressure. Improved characterization of the myeloid compartment may lead to greater insight into the fundamental biology of tumor-immune interactions, novel therapeutic strategies, and other applications. Citation Format: Sushil Kumar, Sarah Warren, Tiziana Cotechini, Terry Medler, Christina Bailey, Joseph M. Beechem, Joseph M. Beechem, Lisa M. Coussens. Validation of human and mouse myeloid panels on the NanoString® nCounter® Platform [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3706. doi:10.1158/1538-7445.AM2017-3706

MP14-07 DEVELOPMENT OF A LIQUID BIOPSY USING EXTRACELLULAR VESICLES TO ASSESS THE SYSTEMIC T CELL IMMUNE LANDSCAPE IN BLADDER CANCER

T cells can have different activities based on receptor expression and cytokine production. Current methods to classify and assess immune cell behavior include flow cytometry and immunohistochemistry, which require immune cell labeling and tissue fixation. A non-invasive method for determining T cell behavior is needed to study immune cell behaviors in tumors and evaluate novel immunotherapies. Activated T cells require high rates of glycolysis to maintain immune activities. Therefore, we are developing optical metabolic imaging (OMI) to assess the metabolic profile of T cell subtypes and activation states using cells isolated from human blood. OMI probes the fluorescence intensity and lifetime of the metabolic coenzymes NAD(P)H and FAD, to quantitate the redox state of the cell through the optical redox ratio (NAD(P)H fluorescence intensity divided by the sum of NAD(P)H and FAD fluorescence intensity) and co-enzyme binding. Our results show that the optical redox ratio is increased in activated populations of unsorted T cells, and of CD8+ T cells, consistently across four different donors. Single-cell analysis of the unsorted, unactivated T cell populations revealed a small portion of cells in an activated state. Inter-donor heterogeneity highlights the variability of immune responses between patients. These results indicate that OMI is a powerful tool for assessing T cell subtype and behavior. OMI utilizes the autofluorescent properties of NAD(P)H and FAD, and thus is contrast agent free, non-damaging, and requires no genetic manipulation. Therefore, OMI can be used to image T cell interactions with tumors in time-course studies of tumor development or assess the efficacy of immunotherapy.

BackgroundRecent research has identified a potential protective effect of higher numbers of circulating lymphocytes on colorectal cancer (CRC) development. However, the importance of different lymphocyte subtypes and activation states in CRC development and the biological pathways driving this relationship remain poorly understood and warrant further investigation. Specifically, CD4+ T cells – a highly dynamic lymphocyte subtype – undergo remodelling upon activation to induce the expression of genes critical for their effector function. Previous studies investigating their role in CRC risk have used bulk tissue, limiting our current understanding of the role of these cells to static, non-dynamic relationships only.MethodsHere, we combined two genetic epidemiological methods – Mendelian randomisation (MR) and genetic colocalisation – to evaluate evidence for causal relationships of gene expression on CRC risk across multiple CD4+ T cell subtypes and activation stage. Genetic proxies were obtained from single-cell transcriptomic data, allowing us to investigate the causal effect of expression of 1,805 genes across five CD4+ T cell activation states on CRC risk (78,473 cases; 107,143 controls). We repeated analyses stratified by CRC anatomical subsites and sex, and performed a sensitivity analysis to evaluate whether the observed effect estimates were likely to be CD4+ T cell-specific.ResultsWe identified six genes with evidence (FDR-P<0.05 in MR analyses and H4>0.8 in genetic colocalisation analyses) for a causal role of CD4+ T cell expression in CRC development – FADS2, FHL3, HLA-DRB1, HLA-DRB5, RPL28, and TMEM258. We observed differences in causal estimates of gene expression on CRC risk across different CD4+ T cell subtypes and activation timepoints, as well as CRC anatomical subsites and sex. However, our sensitivity analysis revealed that the genetic proxies used to instrument gene expression in CD4+ T cells also act as eQTLs in other tissues, highlighting the challenges of using genetic proxies to instrument tissue-specific expression changes.ConclusionsOur study demonstrates the importance of capturing the dynamic nature of CD4+ T cells in understanding disease risk, and prioritises genes for further investigation in cancer prevention research.

Duobody-CD3xCD20 Induces Potent Anti-Tumor Activity in Malignant Lymph Node B Cells from Patients with DLBCL, FL and MCL Ex Vivo, Irrespective of Prior Treatment with CD20 Monoclonal Antibodies

Context It has been demonstrated that dopamine (DA) plays an important role in numerous cellular processes of T cell. Accumulating evidence suggests that the outcomes of T cell treatment with DA is depended on DA concentrations, T cell subtypes and activation states. However, the detail mechanism of DA function on T cell activation or regulatory T cells is largely unclear. Objective This study aims to explore the mechanisms by which DA regulates the activation of CD4+ T cells and the function of Tregs. Materials and methods T cell proliferation was detected using CCK-8, BrdU incorporation assay or eFluor 450 cell labeling assay, and Western blot were used to detect phosphorylation of p65 and Erk. Nuclear translocation of transcription factors including p65, FOXO1 and NFAT1 were observed under laser confocal microscopy. Results Our present study demonstrated that DA (17 µM) can directly promote CD4+ T cells activation through D2-like receptors by enhancing the phosphorylation of p65, also can impair regulatory CD4+ T cells (Tregs) stability and suppressive function through D1- and D2-like receptors by inhibiting the expression of FOXO1 and NFAT1, which are the transcriptional factors of FOXP3, and by suppressing the expression of IL-10 in Tregs. Injection of DA can inhibit tumor growth in vivo. Conclusions These data indicate a critical role for DA in promotion of CD4+ T helper response, this may applicable in tumor treatment in the future.

NO as an autocrine mediator in the apoptosis of activated microglial cells: correlation between activation and apoptosis of microglial cells

Background: Multi-omic single cell studies are revolutionizing knowledge of the tumor immune microenvironment and positioned to detect rare cell subsets that correlate with response to immunotherapy. DNA-barcoded antibody panels enable the unambiguous classification of cell subsets via simultaneous measurement of surface proteins and transcriptome. However, single-cell sequencing performs best on fresh samples while multi-center clinical trials routinely obtain frozen specimens. Peripheral blood mononuclear cells (PBMCs) are commonly used tissues for charactering immune cell types, dynamics, and signaling in human studies. Our group has pioneered analyses of the tumor micro-environment in several cancer types, and also developed genomic capabilities with the resolution of flow cytometry over thousands of markers. We hypothesized that the development of advanced bioinformatics tools and large antibody panels (137 antibodies) can augment the analysis of frozen samples. Methods: Single cell genomics on matched fresh and frozen human PBMCs was performed using the TotalSeq-C human universal cocktail (BioLegend) and the 10x Genomics Chromium single-cell platform. We created bioinformatics pipelines to compare the quality control metrics, isotype matched control performance, and cell subtype analyses for fresh and frozen samples. Advanced visualization and analysis tools were developed to allow interrogation of the data in a flow cytometry paradigm, including complex gating strategies to identify rarer cell subtypes. Results: In terms of library quality, fresh and frozen cells had similar quality control parameters, although mean reads per cell were moderately lower for frozen vs. fresh tissue in both the RNA and antibody arms of the study. RNA and antibody data were integrated, clustered, and annotated for cell type and subtype. Cell type proportions were comparable across fresh and frozen samples. A panel of 7 antibody isotype controls demonstrated limited non-specific binding. Comparison of individual surface proteins across fresh and frozen samples revealed similar distributions in most cases, although signal was attenuated for a small number of epitopes in frozen samples. Our bioinformatics approaches allowed rare cell subtype identification with sensitivity and selectivity rivaling flow cytometry. Conclusions: Multi-modal single cell genomics, including a 137 antibody panel, and novel bioinformatics analytics reveal that fresh and frozen PBMC samples are largely concordant at both the transcriptome and cell-surface protein levels. Our pilot study is being expanded to larger scale clinical settings and may help characterize important cell populations that are associated with disease status, pharmacodynamics, and therapeutic response. Citation Format: Nathan O. Siemers, Jasmine Chen, Parminder Mankoo, Shazia Ilyas. Evaluation of large antibody panels in single-cell genomic immunophenotyping of fresh and preserved human leukocytes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2244.

Two major K(+) channels are expressed in T cells, (i) the voltage-dependent K(V)1.3 channel and (ii) the Ca(2+)-activated K(+) channel KCa 3.1 (IKCa channel). Both critically influence T cell effector functions in vitro and animal models in vivo. Here we identify and characterize TWIK-related acid-sensitive potassium channel 1 (TASK1) and TASK3 as an important third K(+) conductance on T lymphocytes. T lymphocytes constitutively express TASK1 and -3 protein. Application of semi-selective TASK blockers resulted in a significant reduction of cytokine production and cell proliferation. Interference with TASK channels on CD3(+) T cells revealed a dose-dependent reduction ( approximately 40%) of an outward current in patch clamp recordings indicative of TASK channels, a finding confirmed by computational modeling. In vivo relevance of our findings was addressed in an experimental model of multiple sclerosis, adoptive transfer experimental autoimmune encephalomyelitis. Pretreatment of myelin basic protein-specific encephalitogenic T lymphocytes with TASK modulators was associated with significant amelioration of the disease course in Lewis rats. These data introduce K(2)P channels as novel potassium conductance on T lymphocytes critically influencing T cell effector function and identify a possible molecular target for immunomodulation in T cell-mediated autoimmune disorders.